Method for preparing a pharmaceutical composition in the form of microparticles

ABSTRACT

A method for preparing a pharmaceutical composition in the form of microparticles, the composition thus obtained and its use for preparing injectable suspensions.

SUMMARY OF THE INVENTION

More precisely, the object of the invention is a method for preparing apharmaceutical composition which is aimed at providing a prolonged and acontrolled release of a medicamentous substance, which is obtained isthe form of microparticles of a copolymer of lactic and of glycolicacids, and which incorporates, as the active substance, the pamoate,tannate, stearate or palmitate salt of a natural or of a syntheticpeptide and, more particularly, of a peptide comprising 3 to 45 aminoacids.

STATE OF THE ART

Various solutions have been proposed to this day for preparingcompositions ensuring a prolonged and are controlled release ofmedicamentous substances, which are based on the preparation ofbiodegradable implants, on microencapsulation or on the preparation ofporous biodegradable matrixes, for example in the form of microparticlesof various grain sizes. In this respect, one can mention EP-A-0052510for microencapsulation and EP-A-0058481 or U.S. Pat. No. 3,976,071 forthe preparation of implants or of porous biodegradable matrixes. Allthese techniques make use of a preliminary dissolution in an organicsolvent of the biodegradable polymer or copolymer used as support, and,when required, the dissolution of the medicamentous substance itself.Even though the dispersion of the active substance throughout thebiodegradable mass is satisfactory in such cases, problems with traceamounts of residual solvent are always encountered, which may jeopardizethe use of such compositions in therapeutic applications. The selectionof solvents with a low toxicity or the thorough removal of traces ofsolvent can sometimes be complex and expensive, or it can result in anunacceptable loss of purity of the product.

It has also been proposed to dry-mix--i.e. without any solvent--aproteinic substance (Bovine Serum Albumine) and a biodegradablecopolymer of lactic and of glycolic acids in the form of powders, andthen to carry out a compression at the melting temperature of themixture thus obtained (J. D. Gresser and al., Biopolymeric ControlledRelease System Vol. II, p. 136). This technique did not provesatisfactory, in particular with respect to the homogeneity of thedistribution of the proteinic substance (BSA) throughout the mass.

Against all expectations, it was found that these various problems couldbe overcome even when using as starting material the same type ofbiodegradable polymers or copolymers and of natural or syntheticpeptides, such as octa-, nona-, or decapeptides, and more generallypeptides comprising 3 to 45 amino acids, through the application of themethod of the invention.

PREFERRED EMBODIMENTS OF THE INVENTION

According to the invention, natural or synthetic peptides are used inthe form of salts, more precisely as pamoates, tannates, stearates orpalmitates, and preferably as pamoates. It can be noted in this respect,that these salts of peptides are insoluble in water.

The above-mentioned salts, as well as the copolymers of lactic acid (L-or D,L-lactic acid) and of glycolic acid are used in the form of apowder, and more particularly, in the form of microparticles with anaverage grain size below approximately 200 microns. Good results wereobtained with microparticles of copolymer having a grain size in theorder of -80 microns or less, the peptidic salt being capable of havingeven a smaller grain size. The mixture of these materials is carried outby dry-mixing in any appropriate apparatus, for example in a ball mill,and at room temperature (approx. 25° C.) or even at a lower temperature,for example 5° to 10° C. The proportion of the powdered components canvary within a broad range, for example from 0.1 to 15% in weight for thepeptidic salt, depending upon the therapeutic effects required.

According to the invention, once a given mixture is duly homogenized, itis subjected to a progressive compression and, simultaneously, to aprogressive heating, before being extruded. These two operations, aswell as the transport of the mixture to the precompression andpreheating zone can be carried out advantageously using an adequatelydimensioned endless screw. The compression ratio may vary depending onnumerous factors, such as the geometry of the apparatus or the grainsize of the powdered mixture. The control of the preheating and of thechange it undergoes as the mixture progresses is more critical :depending upon the nature of the products to be treated (copolymer,peptide), every endeavour is made to maintain a temperature gradient notexceeding approximately 80° C. The initial temperature to which thepowdered mixture is subjected can be 25° C., lower or higher, dependingon circumstances.

The mixture thus precompressed and preheated is then subjected to anextrusion at a temperature most generally comprised betweenapproximately 80 and 100° C, the upper limit of this range beingdictated by the nature of the medicamentous substance (peptide), whichshould not undergo deterioration. The extrusion can be carried out at apressure which can vary considerably in the range from 50 to 500 kg/cm²,the main point being that the extrusion temperature and pressure beadapted according to the viscosity of the product. Quite obviously, anadequate pressure and an adequate temperature promote the perfecthomogenization of the ingredients and, in particular, the regulardistribution of the peptidic salt throughout the mass of the copolymer.

The actual extrusion is carried out by means of a nozzle of standardshape and dimensions, placed at the downstream end of theabove-mentioned endless screw. The cooling of the extruded product isachieved by any appropriate means, such as cold sterile air or gas orsimply through natural loss of heat.

According to the invention, the extruded product adequately cooled isthen pulverized at low temperature, preferably at a temperature lowerthan 0° C., or even much lower, for example -10° C. or -30° C. It isadvantageous to use cryogenic pulverization, a technique known per se.The product thus pulverized is then subjected to a grading of themicroparticles according to their average grain size, those having agrain size below 200 microns and preferably below or equal to 180microns being retained, in accordance with the method of the invention.The grading of the microparticles can be carried out, for example, bysieving. The graded microparticles are collected and they are then readyfor use.

In accordance with the method of the invention, the steps describedabove take place in succession, without any excessive delay between twosuccessive steps. An advantage of this method is that it can also becarried out as a continuous process, with all the operations takingplace in succession, simply by transferring the treated mixture.

According to the invention, one can use as copolymer of lactic andglycolic acids, any type of biodegradable copolymer comprised of such abase, and preferably, a copolymer of L- or D,L-lactic acid containingrespectively from 45% to 90% (moles) of lactic acid units and 55% to 10%(moles) of glycolic acid units. Such polymers are readily prepared asdescribed in the above-mentioned literature or they can be obtained fromspecialized firms.

The salts of peptides, whether natural or synthetic, thus incorporatedinto the mass of the copolymer, are preferably salts of peptidescomprising from 3 to 45 amino acids and, more particularly, salts ofLH-RH (Luteinizing Hormone - Releasing Hormone), of somatostatin, ofGH-RH (Growth Hormone - Releasing Hormone) or of calcitonin, or of theirsynthetic homologous or analogous.

More particularly, the pamoate of LH-RH, of somatostatin or of one oftheir homologues or analogues selected from ##STR1## (R¹ lower alkyl)are concerned, this list not being limitative.

The microparticles obtained according to the method of the inventionfrom the above-mentioned ingredients are then used, after an appropriatesterilization, for the preparation of injectable suspensions.

The following Examples illustrate the invention in a more detailedmanner, without however limiting its scope.

EXAMPLE 1

20 g of a 50:50 (% moles) copolymer of D,L-lactic and glycolic acids inthe form of granules having a diameter ranging approximately from 3 to 5mm were first milled at low temperature and sieved to obtainmicroparticles having an average grain size of 180 microns or less.

To this powdered mass, were added 0.490g of finely pulverized pamoate ofD-Trp6-LH-RH (formula of the peptide : ##STR2## The product is comprisedof microparticles with a grain size of about 10 microns and it has anamorphous structure. The resulting mixture was homogenized in a mill atroom temperature.

The homogenized mixture was then placed inside an apparatus providedwith an endless screw coupled to a conventional extrusion nozzle. Theendless screw has a length of about 25 cm and a diameter of about 1.5cm. It includes a first zone, the purpose of which is exclusively tomove the mixture, and which neighbours a second zone designed for thecompression and the preheating.

As the mixture travels, it is heated from 25° to approximately 80° C.,the travelling speed being adjusted so that this phase lasts about 5minutes. The actual extrusion takes place at 98° C., through anextrusion nozzle having an opening with a diameter of approximately 1.5mm

The filaments thus obtained are then left to cool at room temperature,cut into short portions and finally milled at -30° C. After sieving,microparticles with an average grain size of 180 microns or less arecollected.

The chemical analysis, carried out on samples of the product afterextrusion and milling, confirms the perfect homogeneity of thedispersion of the active substance throughout the mass of the polymer.

The microparticles obtained above were subjected to a sterilization withgamma rays and then they were suspended in an appropriate sterilevehicle. In vivo tests (determination of the level of blood testosteronein strains of male rats) confirm the regular release of the activesubstance during at least 25 days, which results in a fall oftestosterone to castration levels.

EXAMPLE 2

Microparticles of a 50:50 (% moles) copolymer of D,L-lactic-glycolicacids were prepared according to the procedure of Example 1, to includea comparable level of pamoate of one of the following decapeptides :##STR3## (R¹ =ethyl)

The activity tests carried out in vivo confirm a regular release of theactive substance over several weeks.

EXAMPLE 3

13.85 g of a 75:25 (% moles) copolymer of D,L-lactic and glycolic acidsin the form of granules with a diameter in the order of 3 to 5 mm werefirst milled at low temperature and sieved to obtain microparticles withan average grain size of 180 microns or less.

To this powdered mass, 1.15 g of finely pulverized pamoate of D-Trp⁶-LH-RH (formula of the peptide : ##STR4## were added. The productconsists of microcapsules with a grain size of about 10 microns and ithas an amorphous structure. The resulting mixture was homogenized atroom temperature in a mill, and finally subjected to the treatmentdescribed in Example 1.

After cryopulverizing, sieving and finally sterilizing with gamma rays,the microparticles were suspended in an appropriate sterile vehicle. Invivo tests (determination of the level of blood testosterone in strainsof male rats) confirm the regular release of the active substance duringat least 40 days, which results in a fall of testosterone to castrationlevels.

EXAMPLE 4

The procedure of Example 1 was followed, starting from 18 g of the 50:50(% moles) copolymer of D,L-lactic and glycolic acids and from 2.85 g ofpamoate of an analogue of somatostatin - formula of the peptide :##STR5## to obtain microparticles having the desired grain size.

The chemical analysis carried out on the samples of the product afterextrusion and milling, confirm the perfect homogeneity of the dispersionof the active substance throughout the mass of the copolymer.

In vivo tests further confirm the controlled release of the activesubstance (analogue of somatostatin) over a period of at least 7 days.

EXAMPLE 5

The procedure of Example 4 was repeated, starting this time from 13.50 gof a 75:25 copolymer of D,L-lactic-glycolic acids and from 1.50 g ofpamoate of the above-mentioned analogue of somatostatin.

The microparticles thus obtained, once sterilized by means of gammarays, were finally suspended in an appropriate sterile vehicle. In vivotests (determination of the level of the analogue of somatostatin inblood serum of rats subjected to a single injection at t_(o)) indicate acontrolled release of the active substance during at least 15 days.

EXAMPLE 6

The procedure of Example 4 was used, to obtain microparticles of a 50:50(% moles) copolymer of D,L-lactic-glycolic acids, containing a similaramount of pamoate of one of the following octapeptides : ##STR6##

The chemical analysis carried out on samples of the product afterextrusion and milling, confirms the perfect homogeneity of thedispersion of the active substance throughout the mass of the copolymer.

During the above-described experimentation, it was noted that theextruded filaments, once cut into short rods of an appropriate length,could be used directly as implants, after sterilization. Such implantsensure also a prolonged and a controlled release of the activesubstance.

What I claim is:
 1. A method for preparing a pharmaceutical compositionwhich is aimed at providing a prolonged and a controlled release ofmedicamentous substance, which is obtained in the form of microparticlesof a copolymer of lactic and glycolic acids, and which incorporates, asthe active substance, the pamoate, tannate, stearate or palmitate saltof a natural or of a synthetic peptide, characterized in that:a) theselected copolymer and active substance, both present in the form ofmicroparticles whose average grain size is below approximately 200microns, are dry-mixed in relative amounts such that the activesubstance is present in a proportion of about 0.1-15% by weight; b) thepowdered mixture thus obtained is compressed and is heated up toapproximately 80° C.; c) the precompressed and preheated mixture issubjected to an extrusion at a temperature of between approximately 80°and 100° C.; and d) the product resulting from the extrusion ispulverized at low temperature and then, the microparticles having agrain size below approximately 200 microns are selected and finallycollected.
 2. A method according to claim 1, characterized in that themicroparticles of copolymer have an average grain size below or equal to180 microns.
 3. A method according to claim 2, characterized in that theprecompression and the preheating of the mixture are carried outsimultaneously by means of an endless screw.
 4. A method according toclaim 3, characterized in that the extrusion is carried out at apressure in the range from 50 to 500 kg/cm².
 5. A method according toclaim 4, characterized in that the pulverization of the productresulting from the extrusion is a cryogenic pulverization.
 6. A methodaccording to claim 5, characterized in that the selection of themicroparticles resulting from the pulverization is carried out bysieving.
 7. A method according to claim 6, characterized in that thecopolymer of lactic and glycolic acids is a copolymer of L- orD,L-lactic acid containing respectively 45 to 90% (moles) of lactic acidunits and 55 to 10% (moles) of glycolic acid units.
 8. A methodaccording to claim 7, characterized in that the active substance is thepamoate, tannate, stearate or palmitate of a natural or of a syntheticpeptide comprising 3 to 45 amino acids, and including LH-RH,somatostatin, GH-RH, calcitonin or one of their synthetic analogues orhomologues.
 9. A method according to claim 8, characterized in that theactive substance is a pamoate of LH-RH, of somatostatin or of one oftheir analogues or homologues selected from ##STR7## (R¹ =lower alkyl).10. A method for preparing a pharmaceutical composition which provides aprolonged and controlled release of an active substance, whichcomprises:a) mixing microparticles of a copolymer of lactic and glycolicacids with microparticles of an active substance of an insoluble salt ofa natural or synthetic peptide comprising 3 to 45 amino acids for asufficient time to form a homogenous mixture containing about 0.1 to 15%by weight of the active substance, each of said microparticles having anaverage grain size of below approximately 200 microns; and b)precompressing and preheating the mixture prior to extruding theprecompressed and preheated mixture at a temperature of betweenapproximately 80° and 100° C. to form a product for use as saidpharmaceutical composition.
 11. A method according to claim 10 whichfurther comprises:c) pulverizing the extruded product at lowtemperature; and d) selecting the microparticles which have a grain sizeof below about 200 microns for use as said pharmaceutical composition.12. A method according to claim 11 which further comprises selecting themicroparticles of the copolymer to have an average grain size of 180microns or less.
 13. A method according to claim 11 wherein theprecompressing and preheating of the mixture are carried outsimultaneously by passing the mixture through an endless screw.
 14. Amethod according to claim 11 wherein the extrusion is carried out at apressure in the range of about 50 to 500 kg/cm².
 15. A method accordingto claim 11 which further comprises pulverizing the extruded product atcryogenic temperatures.
 16. A method according to claim 11 wherein theselection of the microparticles for use as the pharmaceuticalcomposition is obtained by sieving the desired grain size of thepulverized product.
 17. A method according to claim 11 which furthercomprises selecting the copolymer of lactic and glycolic acids to be acopolymer of L- or D,L-lactic acid containing 45 to 90% (moles) oflactic acid units and 55 to 10% (moles) of glycolic acid units.
 18. Amethod according to claim 11 which further comprises selecting theactive substance to be a pamoate, tannate, stearate or palmitate salt ofLH-RH, somatostatin, GH-RH, calcitonin or a synthetic analogue orhomologue thereof.
 19. A method according to claim 11 which furthercomprises selecting the active substance to be a pamoate salt of LH-RH,somatostatin or an analogue or homologue thereof selected from the groupconsisting of: ##STR8## where R¹ =a lower alkyl group.
 20. The method ofclaim 1 wherein additional heat and pressure are simultaneously appliedin slowly increasing amounts to the powdered mixture of the selectedcopolymer and active substance.
 21. The method of claim 20 whereinadditional heat and pressure are simultaneously applied in slowlyincreasing amounts to the powdered mixture of the selected copolymer andactive substance as the mixture is transported in an endless screw. 22.The method of claim 10 wherein additional heat and pressure aresimultaneously applied in slowly increasing amounts to the powderedmixture of the selected copolymer and active substance.
 23. The methodof claim 22 wherein additional heat and pressure are simultaneouslyapplied in slowly increasing amounts to the powdered mixture of theselected copolymer and active substance as the mixture is transported inan endless screw.